Pavel Pisa

CD4+CD25high T Cells Are Enriched in the Tumor and Peripheral Blood of Prostate Cancer Patients

In this study, we investigated whether CD4+CD25high regulatory T cells (Treg) are increased in the tumor tissue and peripheral blood of early-stage prostate cancer patients undergoing prostatectomy. We show that the prevalence of CD4+CD25high T cells inside the prostate was significantly higher in the tumor compared with benign tissue from the same prostate. Furthermore, the frequency of CD4+CD25high T cells in peripheral blood was significantly higher in prostate cancer patients compared with normal donors. A proportion of the CD4+CD25high T cells was also shown to be glucocorticoid-induced TNF receptor, ICOS, and FOXP3 positive. Moreover, CD4+CD25+ T cells from blood and supernatants from cultured prostate tumor tissue samples exhibited immunosuppressive function in vitro. Furthermore, supernatants from cultured prostate tissue samples and prostate cancer ascites fluid induced migration of CD4+CD25+ T cells and were shown to contain the regulatory T cell chemokine CCL22 by ELISA. Our findings indicate that Tregs are an important cellular component of early-stage prostate tumors, and thus new therapeutic strategies aimed at inhibition or depletion of Tregs may improve prostate cancer immunotherapy.

Tumor-induced immune dysfunction.

Abstract Immune system-based approaches for the treatment of malignant disease over the past decades have often focused on cytolytic effector cells such as cytotoxic T lymphocytes (CTL), and natural killer (NK) cells. It has also been demonstrated that tumor-bearing mice can be cured using a wide variety of approaches, some of which involve cytokine-mediated enhancement of CTL and NK cell activity. However, the apparent success in mice stands in contrast to the current situation in the clinic, wherein only a minority of patients have thus far benefited from CTL- or NK cell-based antitumor approaches. The underlying causes of tumor-associated immune suppression of CTL and NK cell activity are discussed, and features of interest shared with HIV infection, leprosy, and rheumatoid arthritis are also be mentioned. Remarkable and very recent observations have shed more light upon the causes of dysfunctional alterations in CTL and NK cells often associated with these diseases, that in turn have suggested new immunotherapeutic approaches for cancer and infectious disease.

Downregulation of telomerase reverse transcriptase mRNA expression by wild type p53 in human tumor cells.

The p53 tumor suppressor protein inhibits the formation of tumors through induction of cell cycle arrest and/or apoptosis. In the present study we demonstrated that p53 is also a powerful inhibitor of human telomerase reverse transcriptase (hTERT), a key component for telomerase. Activation of either exogenous temperature-sensitive (ts) p53 in BL41 Burkitt lymphoma cells or endogenous wild type (wt) p53 at a physiological level in MCF-7 breast carcinoma cells triggered a rapid downregulation of hTERT mRNA expression, independently of the induction of the p53 target gene p21. Co-transfection of an hTERT promoter construct with wt p53 but not mutant p53 in HeLa cells inhibited the hTERT promoter activity. Furthermore, the activation of the hTERT promoter in Drosophila Schneider SL2 cells was completely dependent on the ectopic expression of Sp1 and was abrogated by wt p53. Finally, wt p53 inhibited Sp1 binding to the hTERT proximal promoter by forming a p53-Sp1 complex. Since activation of telomerase, widely observed in human tumor cell lines and primary tumors, is a critical step in tumorigenesis, wt p53-triggered inhibition of hTERT/telomerase expression may reflect yet another mechanism of p53-mediated tumor suppression. Our findings provide new insights into both the biological function of p53 and the regulation of hTERT/telomerase expression.

A phase I trial of DNA vaccination with a plasmid expressing prostate-specific antigen in patients with hormone-refractory prostate cancer.

Prostate-specific antigen (PSA) is a serine protease secreted at low levels by normal luminal epithelial cells of the prostate and in significantly higher levels by prostate cancer cells. Therefore, PSA is a potential target for various immunotherapeutical approaches against prostate cancer. DNA vaccination has been investigated as immunotherapy for infectious diseases in patients and for specific treatment of cancer in certain animal models. In animal studies, we have demonstrated that vaccination with plasmid vector pVAX/PSA results in PSA-specific cellular response and protection against tumour challenge. The purpose of the trial was to evaluate the safety, feasibility and biological efficacy of pVAX/PSA vaccine in the clinic. A phase I trial of pVAX/PSA, together with cytokine granulocyte/macrophage-colony stimulating factor (GM-CSF) (Molgramostim) and IL-2 (Aldesleukin) as vaccine adjuvants, was carried out in patients with hormone-refractory prostate cancer. To evaluate the biologically active dose, the vaccine was administered during five cycles in doses of 100, 300 and 900 μg, with three patients in each cohort. Eight patients were evaluable. A PSA-specific cellular immune response, measured by IFN-γ production against recombinant PSA protein, and a rise in anti-PSA IgG were detected in two of three patients after vaccination in the highest dose cohort. A decrease in the slope of PSA was observed in the two patients exhibiting IFN-γ production to PSA. No adverse effects (WHO grade >2) were observed in any dose cohort. We demonstrate that DNA vaccination with a PSA-coding plasmid vector, given with GM-CSF and IL-2 to patients with prostate cancer, is safe and in doses of 900 μg the vaccine can induce cellular and humoral immune responses against PSA protein.

Skin electroporation: effects on transgene expression, DNA persistence and local tissue environment.

Background Electrical pulses have been used to enhance uptake of molecules into living cells for decades. This technique, often referred to as electroporation, has become an increasingly popular method to enhance in vivo DNA delivery for both gene therapy applications as well as for delivery of vaccines against both infectious diseases and cancer. In vivo electrovaccination (gene delivery followed by electroporation) is currently being investigated in several clinical trials, including DNA delivery to healthy volunteers. However, the mode of action at molecular level is not yet fully understood. Methodology/Principal Findings This study investigates intradermal DNA electrovaccination in detail and describes the effects on expression of the vaccine antigen, plasmid persistence and the local tissue environment. Gene profiling of the vaccination site showed that the combination of DNA and electroporation induced a significant up-regulation of pro-inflammatory genes. In vivo imaging of luciferase activity after electrovaccination demonstrated a rapid onset (minutes) and a long duration (months) of transgene expression. However, when the more immunogenic prostate specific antigen (PSA) was co-administered, PSA-specific T cells were induced and concurrently the luciferase expression became undetectable. Electroporation did not affect the long-term persistence of the PSA-expressing plasmid. Conclusions/Significance This study provides important insights to how DNA delivery by intradermal electrovaccination affects the local immunological responses of the skin, transgene expression and clearance of the plasmid. As the described vaccination approach is currently being evaluated in clinical trials, the data provided will be of high significance.

Telomerase activity and the expression of telomerase components in acute myelogenous leukaemia

In 95 leukaemic cell samples from 66 patients with acute myelogenous leukaemia (AML) (47 de novo and 19 secondary AML) telomerase activity was determined and the expression of the telomerase components: telomerase reverse transcriptase (hTERT), telomerase RNA template (hTR) and telomerase‐associated protein (TP1) evaluated by RT‐PCR. Compared to peripheral blood mononuclear cells (PBMC) from normal adult 87% (82/95) of patient samples exhibited elevated telomerase activity. hTERT, but not hTR and TP1 expression strongly correlated with the levels of telomerase activity (r = 0.47, P < 0.0001). The levels of telomerase activity were significantly higher at time of relapse or progression than at time of diagnosis (P = 0.003), and correlated to CD34 expression and chromosomal abnormalities of leukaemic cells (P = 0.01 and P = 0.001 respectively). The rate and duration of complete remission (CR) did not correlate with the levels of telomerase activity at diagnosis. Among eight patients in first relapse, however, two of three with low levels of telomerase activity re‐entered CR, whereas none of five patients with high telomerase activity achieved a second CR. Taken together, telomerase activation/up‐regulation in AML is a disease progression‐associated event. Undifferentiated status and chromosomal aberration also lead to the up‐regulation of telomerase activity in AML.

Optimization of Skin Electroporation in Mice to Increase Tolerability of DNA Vaccine Delivery to Patients

Electroporation has, during the last years, proven to be a very successful delivery method for DNA vaccines and has now reached clinical evaluation. Although intramuscular electroporation is practical in animal models, intradermal electroporation might be more suitable for clinical administration. Skin is the most accessible organ of the body and has professional antigen-presenting cells in large amounts; thus, skin is an ideal target for DNA vaccine delivery. Moreover, intradermal electroporation has clear clinical benefits such as improved safety and tolerability. This article describes improvements for effective and tolerable DNA delivery to skin. The time of pulse delivery has been shortened by 90% and even pulse programs of 240-ms total duration generate robust immune responses. We show that a single vaccination using an optimized gene delivery generates (i) high and consistent protein expression in vivo, (ii) cytotoxic antigen-specific T cells expressing both IFNgamma and CD107a (lysosomal-associated membrane protein 1). Furthermore, application of a topical anesthetic cream prior to vaccination does not affect the number or function of the antigen-specific T cells induced. This suggests that local anesthesia can be used to further decrease the sensation of pulse delivery in patients.

Tumor escape mechanisms in prostate cancer

Numerous immunotherapy trials have been carried out in prostate cancer (PC) patients, with induction of antigen-specific T cells in some cases. Despite this capability, limited success is seen in terms of tumor regression or survival. In this review, we discuss the evidence for tumor escape strategies that may contribute to vaccine failure in the setting of PC. These include defects in antigen presentation, production of immunosuppressive substances, induction of T cell death, T cell receptor dysfunction, and the presence of tolerogenic dendritic cells and regulatory T cells inside prostate tumors. It is clear that novel strategies aimed at preventing tumor escape, such as small molecular weight inhibitors of immunosuppressive molecules, adoptive transfer of TCR transgenic T cells, removal of Tregs, combined with anti-androgen therapy and prostate-specific vaccines, need to be examined further in PC patients.

Immune monitoring in a phase 1 trial of a PSA DNA vaccine in patients with hormone-refractory prostate cancer

Prostate cancer remains a leading cause of cancer illness and death among men in Europe. No curative treatment exists when the disease has spread beyond the prostate. Immunotherapy with DNA vaccines has emerged as a potential therapeutic approach for the induction of antitumor specific cytotoxic T lymphocytes. In this study six patients with hormone-refractory prostate cancer were monitored for their ability to mount PSA-specific cellular responses after receiving a pVAX/PSA DNA vaccine (patients 1-3, 100 μg; patients 7-9, 900 μg) with recombinant GM-CSF and IL-2 as adjuvants. IFNγ ELISPOT showed that naturally processed PSA protein and PSA peptides are recognized by T cells in the blood of some prostate cancer patients after a PSA DNA vaccine. Analysis of other cytokines showed the production of IL-4 and IL-6 but importantly did not show an increase in the number of IL-10-producing cells after vaccination in any of the patients. The authors conclude that a pVAX/PSA DNA vaccine can induce PSA-specific cellular immune responses in patients with hormone-refractory prostate cancer, thus emphasizing the potential for PSA as a target molecule for the immunotherapy of prostate cancer.

Tumor-Specific Bacteriophages Induce Tumor Destruction through Activation of Tumor-Associated Macrophages

We recently reported that administration of tumor-specific bacteriophages initiates infiltration of neutrophilic granulocytes with subsequent regression of established B16 tumors. The aim of the current study was to investigate the mechanism of action of bacteriophage-induced tumor regression and to examine possible stimulatory effects of bacteriophages on macrophages. We observed that the mechanism of phage-induced tumor regression is TLR dependent as no signs of tumor destruction or neutrophil infiltration were observed in tumors in MyD88−/− mice in which TLR signaling is abolished. The microenvironment of bacteriophage-treated tumors was further analyzed by gene profiling through applying a low-density array preferentially designed to detect genes expressed by activated APCs, which demonstrated that the M2-polarized tumor microenvironment switched to a more M1-polarized milieu following phage treatment. Bacteriophage stimulation induced secretion of proinflammatory cytokines in both normal mouse macrophages and tumor-associated macrophages (TAMs) and increased expression of molecules involved in Ag presentation and costimulation. Furthermore, mouse neutrophils selectively migrated toward mediators secreted by bacteriophage-stimulated TAMs. Under these conditions, the neutrophils also exhibited increased cytotoxicity toward B16 mouse melanoma target cells. These results describe a close interplay of the innate immune system in which bacteriophages, located to the tumor microenvironment due to their specificity, stimulate TAMs to secrete factors that promote recruitment of neutrophils and potentiate neutrophil-mediated tumor destruction.